Coronaviruses are emerging, evolving and newly discovered viruses that are posing a threat to public health. Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. NL63-CoV was recently identified as a second coronavirus associated with respiratory illness. Like SARS-CoV, NL63-CoV enters its target cell via angiotensin-converting enzyme 2. Unlike SARS-CoV, the mortality rate of people infected with NL63-CoV is higher in children. To combat the effects of these viruses, the development of antivirals is warranted. A logical target for antiviral drug development against these coronaviruses is the 3C-like protease (3CLpro). This enzyme is essential for viral replication, and has become the focus of numerous drug discovery efforts worldwide. Evaluation of inhibitory activity of several published lead compounds has proven limited due to cytotoxicity issues, loss of activity in the presence of reducing agent, or lack of specificity. We propose to identify novel chemical probes that inhibit SARS-3CLpro and NL63-3CLpro by performing high throughput screening (HTS) in collaboration with the Molecular Libraries Screening Centers Network (MLSCN). A highly sensitive assay was developed in our laboratory for routine analysis and high-throughput screening. The assay is a Fvrster resonance energy transfer (FRET) based assay that uses a peptide substrate labeled with HiLyte fluorTM488 and QXLTM520. The QXLTM520 quenches the fluorescence of HiLyte fluorTM488 that is released upon peptide cleavage. The assay provides a high Z-factor and is available for measuring 3CLpro activity from both SARS- 3CLpro and NL63-3CLpro. The primary FRET based assay for identification of novel chemical probes will be followed by secondary screening to determine IC50 values for lead probes. PUBLIC HEALTH RELEVANCE: Infectious diseases remain among the leading causes of death worldwide. The coronaviruses SARS-CoV and NL63-CoV are infectious agents responsible for life-threatening respiratory syndromes. Discovery of chemical probes and antivirals to confront these diseases remain a priority for public health. [unreadable] [unreadable] [unreadable]